Methods and devices for non-therman polymerase chain reaction

ABSTRACT

Methods and devices for non-thermal PCR amplification of nucleic acid sequences. An electrical potential is applied to cause non-thermal separation of strands of a double-stranded nucleic acid or double-stranded nucleic acid/primer extension product.

FIELD OF THE INVENTION

The present invention relates generally to methods and devices useablefor analysis of deoxyribonucleic acid (DNA) and more particularly todevices and methods for denaturing nucleic acid products such as duringperformance of a polymerase chain reaction (PCR) analysis.

BACKGROUND OF THE INVENTION

Pursuant to 37 CFR 1.71(e), this patent document contains material whichis subject to copyright protection. The copyright owner has no objectionto facsimile reproduction of the entire patent document or the patentdisclosure, as it appears in the Patent and Trademark Office patent fileor records, but otherwise reserves all copyright rights whatsoever.

Deoxyribonucleic acid (DNA) molecules carry the genetic code thatdetermines many physical and functional characteristics of organisms.Each DNA molecule consists of two helical backbones or “strands.” Thehelical strands are bound to each other to form a double helix. Each DNAstrand is made up of four types of nucleotide bases, namely, adenine,guanine, thiamine and cytosine. The nucleotide bases on one strand formgeometrically specific bonds (hydrogen bonds) with nucleotide the sametype on the adjacent strand. Thus, the bases bond in type-specificpairs, i.e., adenine to adenine, guanine to guanine, etc. If the strandsare subsequently separated, each strand provides a pattern of successivebases that can be used as a template for reconstruction of a DNAmolecule that is the same as the original DNA molecule. This processoccurs naturally in living organisms and is known as “replication.”

Modern DNA analysis techniques are frequently used to identify, match orcharacterize DNA by determining the specific nucleotide sequence thatexists in DNA sample. However, sometimes, the available DNA sample isrelatively small—such as when the DNA originates from a microorganism ortiny drop of body fluid found at a crime scene. When the available DNAsample is small, it is necessary to amplify the DNA from the sample.This amplification is performed by a laboratory technique that, in manyrespects, mimics natural DNA replication. This laboratory technique isknown as “polymerase chain reaction” (PCR). U.S. Pat. Nos. 4,683,202 and4,683,195 describe basic PCR technology and the entire disclosures ofboth such patents are expressly incorporated herein by reference.

PCR is typically carried out in cycles, with each cycle consisting ofthree steps, namely denaturation, annealing and extension. Prior to thedenaturation step, a sample mixture is typically prepared which containsthe DNA (or a quantity of body fluid or tissue that is believed tocontain the DNA), primers and enzyme(s).

During the denaturation step, all or substantially all (e.g., 99%) ofthe DNA double helix is separated into two individual complimentarystrands. This has heretofore been done by heating a DNA sample mixtureto a temperature of between about 90 and 105 degrees C. for a period ofbetween about one and ten minutes. This elevated temperature causesthermal separation of the DNA double. In some applications, thisincrease in temperature can also serve to terminate chemical reactionswhich had begun within the sample during a previous cycle.

In the annealing step, the specific oligonucleotide primers are attachedto the DNA strands. Primers are needed because the DNA enzymes cannotstart DNA chains from scratch. Instead, the primer is required todetermine the location along a particular DNA template at which thesynthesis of the complementary strand will begin. This allows thetechnique to be used to amplify a particular target region of DNA byselection of primers that are specific to that target region. Inessence, each primer is a synthetic segment of single-stranded DNA thatcontains about 20-30 bases and a chemical label which allows it to belocated and identified. In most PCR procedures, two primers are used,one for each of the complementary single DNA strands produced during thedenaturation step. The annealing step is typically accomplished bylowering the temperature to between about 50 and 60.degrees C. Thiscauses the primers to attach to the individual DNA strands.

Once the annealing step has caused the primers to bind to the DNAstrands, the temperature is again raised, typically to greater than 70degrees C., causing the enzyme(s) to activate. This activation of theenzyme(s) causes replication of the DNA strands. More specifically, theenzyme(s) synthesizes new double-stranded DNA molecules by facilitatingthe joining of complementary nucleotides (i.e., the sugar joined to abase and to a phosphate group) in the sample mixture.

As a result, at the end of the first PCR cycle, two new DNA strands arepresent, each of which is identical to the original target DNA strandthat was denatured and primed. Typically, about 30 of these thermal PCRcycles are required to provide a sufficient amount of DNA for analysis.This can be quite time consuming. For example, each thermal denaturationstep can take about two minutes. Each annealing step can also take abouttwo minutes. Then, each extension step can also take about two minutes.As a result, the thermal cycling in each PCR cycle may takes about sixminutes and a full 30 cycle PCR amplification can take about 3 hours.

Moreover, in thermal PCR, if the temperatures and times of the cyclesare not precisely controlled, the desired amplification may not beachieved. Furthermore, because thermal denaturing requires relativelyhigh temperature, the enzyme(s) in the DNA sample mixture must beselected from a limited number of enzymes that remain stable at theseelevated denaturation temperatures or, alternatively, additional enzymemust be added after each PCR cycle. In this regard, Taq polymerase isthe enzyme frequently used in thermal PCR because it remains stable anddoes not break down at the DNA denaturation temperatures. However, hightemperature enzymes such as Taq lack capabilities that may be availablewith other less temperature-stable enzymes. For example, Taq polymeraselacks a 3′ to 5′ exonuclease activity which, if present, allows theenzyme to identify misplaced bases and replace them with correct basesin the correct positions. The use of an enzyme that has 3′ to 5′exonuclease activity can avoid potentially undesirable amplification oferrors in the target sequence.

Also, thermal PCR is routinely limited to target sequences within acertain size range—e.g., between about 2000 and 3000 base pairs. The useof thermal PCR for amplification of larger targets (e.g., up to 50,000base pairs) can require very long heating cycles and special enzymesthat may not be stable in such long heating cycles.

PCR amplification has a wide range of clinical and investigationalapplications. For example, in subjects who are infected with a specificmicroorganism (e.g., a virus, bacterium, fungus, etc.), a sample of bodyfluid or tissue may contain such a small amount the infecting organism'sDNA that direct identification of the organism's DNA is difficult orimpossible. However, PCR can be used to amplify the infecting organism'sDNA to provide an amount that can be easily analyzed and identified. Forthis reason, PCR is used in a number of diagnostic tests for infectiousdiseases. Additionally, in some instances, PCR techniques can be used tonot only determine that a particular organism is present but to alsoquantify how much of the infecting organism is present in the fluid ortissue sample. Such quantification can be valuable in assessing theseverity of the infection and/or the efficacy of an ongoing treatment(e.g., periodic measurement of viral load in individuals receivingantiviral therapy).

PCR is also used to facilitate testing and screening of donated bloodfor the presence of presence of even very low levels of infectiousorganisms (e.g., Hepatitis B virus (HBV); Hepatitis C virus (HCV 3.0);Human Immunodeficiency viruses Types 1 and 2 (HIV 1,2); HumanT-Lymphotropic virus (HTLV-I/II); Syphilis (Treponema pallidum); WestNile virus (WNV) and Chagas disease (T. cruzi)

Moreover, PCR can be used in genetic counseling. For example, PCRtechniques can be used to analyze small samples of a subject's blood forthe presence or absence of certain genes (e.g., certain specific nucleicacid sequences), thereby indicating whether that subject is predisposedtoward a particular disease or condition and/or predicting how thatsubject may respond to a particular drug or biologic treatment.

Additionally, PCR amplification has also been used to purify aDNA-containing sample or material. In such applications, PCRamplification is used to increase the amount of DNA in the sample ormaterial until the proportional amount of the DNA in the sample ormaterial far exceeds the amounts of contaminants, thus effectivelyreducing the proportional amounts of the contaminants to trivial levels.

Also, PCR amplification can also be used to create DNA libraries thatare useable in conjunction with combinatorial chemistry techniques, forvarious clinical and investigational purposes.

In view of the wide range of potential applications for PCRamplification techniques and the inherent limitations and drawbacksassociated with thermal PCR, the prior art has included various methodsand devices which purportedly eliminate the need for thermal cyclingduring PCR or, at least, provide for faster thermal cycling to limit thetime required for the process.

For example, United Kingdom Patent Application Publication No.GB2,247,880 (Stanley) describes non-thermal methods and apparatus forconverting a double stranded nucleic acid to a denatured single strandednucleic acid by applying an electric potential to a solution containingthe nucleic acid. The described process may be carried out at ambient ornear ambient temperature. The strand separation comes about by electrontransfer to DNA that is free in the solution and adsorbed onto anelectrode. In the examples provided, the solution containing the DNAalso contains a mediator which receives electrons from the electrode andtransfers them to the DNA to effect said strand separation. Suchmediator is defined as “an inorganic or organic molecule which iscapable of reversible electron transfer at an electrode and which passeselectrons on to or receives electrons from a biological molecule, inthis instance the nucleic acid present in solution.” It is furtherstated that the mediator “should be soluble in the solvent for the DNA(which may be water or a solvent other than water) and compounds havinga redox potential of 0 to −2 volts, 25 preferably −0.2 to −1 volt andespecially about −0.4 volts are preferred. Thus the mediator may be awater or solvent soluble compound having conjugated or aromatic groupsand one or more hetero-atoms and may be a compound of the quinone orbipyridyl series, especially a viologen such as 5 methyl viologen or asalt thereof. The choice of mediator is not believed to be criticalprovided that its redox potential is within the required voltage rangeand compound does not otherwise affect or interfere with DNA or othermaterials present in 10 the solution such as enzymes or oligonucleotideprobes. The use of a mediator enables the DNA or other nucleic acidmaterial to be denatured into its individual strands at an appliedvoltage of −0.1 volt or less. Although denaturation has been observed bythe present inventors at a voltage of −1 volt, it is believed that thismay be an overvoltage and the voltage needed to bring about actualdenaturation may be as low as −0.8 volts especially since the redoxpotential of the mediator is 20 typically 0.4 volts.” In someembodiments, the process may be carried out using a modified electrode“in which the electron transfer is e.g. by an electron donating orelectron accepting compound such as a mediator coated onto, or adsorbedonto, the surface of the electrode which is otherwise of an inertmaterial.” Or, “[t]he electron transfer may also be from or to anelectrode consisting at least partially of a mediator compound e.g.formed wholly of the mediator compound.”

U.S. Pat. No. 6,365,400 (Stanley) describes a process for denaturingdouble-stranded nucleic acid material into its individual strands usingan electrochemical cell. This process is an electrical treatment of thenucleic acid with a voltage applied to the nucleic acid material by anelectrode. This process may be used in the detection of nucleic acid byhybridizing with a labeled probe or in the amplification of DNA by apolymerase chain reaction or ligase chain reaction. The process may alsoemploy a promoter compound, such as methyl viologen, to speeddenaturation.

PCT International Patent Publication WO9802573 (Purvis) also describesnon-thermal methods and apparatus for converting a double strandednucleic acid to a denatured single stranded nucleic acid by applying anelectric potential to a solution containing the nucleic acid. Aneffective concentration of Lithium ions are added to the sample to actas a promoter of the denaturation.

United States Patent Application Publication No. 2011/0212492 (Hirahara)describes methods and devices wherein a PCR reaction solution is passedbetween electrodes and low voltage electrical current is passed throughthe reaction solution to generate sufficient Joule heat for the PCRcycle without electrolyzing the reaction solution. This method does usethermal cycling. The low voltage electrical current is merely used toprecisely control the heating of the reaction solution.

Others have described the use of ultrasonic energy, rather than thermalcycling, in PCR. Specifically, United States Patent ApplicationPublication No. 2008/002038 (Patno, et al.) describes a method andapparatus for processing a DNA or RNA sample within a sample processingmodule. The method includes the steps of providing a sample well withinthe sample processing module that contains the DNA or RNA sample,coupling ultrasonic energy from an external source into the samplingwell and denaturing and fragmenting the DNA or RNA sample using theultrasonic energy.

Also, microwave assistance has been described as a means for reducingthe amount of thermal cycling required during a PCR process.Specifically, U.S. Pat. No. 7,537,917 describes a method of microwaveassisted nucleic acid amplification by PCR in which at least thedenaturing and extension steps are carried out under the influence ofmicrowave radiation, while preventing the temperature of the sample fromvarying more than 40° C. from start to finish, and while maintaining thetemperature of the sample from start to finish at no more than 60° C.

Additionally, United States Patent Application Publication No.2003/0104466 (Knapp et al.) describes a non-thermal polymerase chainreaction method that is performed using a microfluidic device. Themicrofluidic device has a reaction chamber or channel that contains atarget nucleic acid sequence and primer sequences, a source of achemical denaturant and a source of polymerase enzyme fluidly connectedto the reaction chamber or channel and a fluid direction system fordelivering the chemical denaturant or the polymerase enzyme to thereaction chamber or channel. Complementary strands of the target nucleicacid sequence are “melted” by delivering a volume of the chemicaldenaturant to the reaction chamber or channel. The primer sequences areannealed to the target nucleic acid by eliminating a denaturing effectof the chemical denaturant. The primer sequences are then extended alongthe target nucleic acid sequence by delivering a volume of thepolymerase enzyme to the reaction chamber or channel. These steps ofmelting, annealing and extending are repeated to amplify the targetnucleic acid sequence.

The entire disclosure of each of the above-cited patents and publishedpatent applications is expressly incorporated herein by reference.

There remains a need in the art for the development of furtheralternatives and improvements to the PCR and nucleic acid amplificationtechniques of the prior art to lessen the time required and/or thecomplexity of the process.

SUMMARY OF THE INVENTIONS

The present invention provides methods and devices for non-thermal PCRamplification of nucleic acid sequences as described herebelow and asshown in the accompanying drawings.

In general, the present invention provides methods and devices forseparating the strands of a double-stranded nucleic acid ordouble-stranded nucleic acid/primer extension product by applying anelectrical potential to cause non-thermal separation of strands of thedouble-stranded nucleic acid or double-stranded nucleic acid/primerextension product, wherein the electrical potential is applied in thesubstantial absence of lithium ions or any initiator or promotersubstance that a) receives electrons from an electrode and transfersthem to the double-stranded nucleic acid or double-stranded nucleic acidprimer extension product and b) has a redox potential of 0 to −2 volts.

In some embodiments, the present invention is used to accomplish thedenaturation step of a PCR process by voltage cycling rather thanthermal cycling. The voltage cycling PCR process of the presentinvention is sometimes referred to herein by the acronym “vcPCR.” Insome applications of vcPCR, the electrical potential is created betweentwo electrodes and one or more oligonucleotide primers is/are attachedto a substrate that comprises or is situated close to an electrode,thereby facilitating the process of the present invention.

Further aspects, details and examples of the present inventions will beunderstood upon reading of the detailed description and examples setforth herebelow.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description and examples are provided for thepurpose of non-exhaustively describing some, but not necessarily all,examples or embodiments of the invention, and shall not limit the scopeof the invention in any way.

FIG. 1 is a flow diagram showing a general method for nucleic acidamplification in accordance with the present invention.

FIG. 2 is a flow diagram showing one example of a method for DNAanalysis in accordance with the present invention.

FIG. 3 (Panels 1 through 8) are schematic diagrams showing a prior artmethod for DNA analysis by solid state bridge amplification in a flowcell.

FIG. 3 (Panels 1 through 8) are schematic diagrams showing steps in aprior art method for DNA analysis by solid state bridge amplification ina flow cell.

FIG. 4 (Panels 1 through 8) are schematic diagrams showing steps in anon-thermal method for DNA analysis by solid state bridge amplificationin a flow cell in accordance with the present invention.

FIG. 5 (Panels 1 through 8) are schematic diagrams showing steps in anon-thermal method for DNA analysis by solid phase amplification with asingle forward primer in accordance with the present invention.

FIG. 6 shows various views of a voltage cycled PCR device of the presentinvention having rectangular sample wells.

FIG. 7 shows various views of a voltage cycled PCR device of the presentinvention having round sample wells.

FIG. 8 shows fluorescence from SYBR Green intercalating dye after 40cycles of voltage cycled PCR in accordance with the present invention,thus indicating that bridge amplification has taken place.

DETAILED DESCRIPTION OF THE INVENTION

The following detailed description and the accompanying drawings towhich it refers are intended to describe some, but not necessarily all,examples or embodiments of the invention. The described examples orembodiments are to be considered, in all respects, as illustrative butnot restrictive. The contents of this detailed description and theaccompanying drawings do not limit the scope of the invention in anyway.

The vcPCR methods of the present invention include various embodiments.For example, described below are vcPCR Bridge Amplification and vcPCRSolid Phase Amplification Using a Single Forward Primer. Also describedbelow are examples of some devices that may be used for performing vcPCRin accordance with the methods of the present invention. In the examplesdescribed below, vcPCR may rely generally on the following features:

-   -   1) At least one of the primers (forward or reverse) is attached        (e.g., by its 5′ end) onto a first electrode (e.g., a “base        electrode”).    -   2) A second electrode (e.g., a counter-electrode) is positioned        close enough to the first electrode to apply a denaturing        electrical potential (e.g., a “melting field”) across the        nucleic acid being denatured.        It has been reported that 65 pN is the critical force at which        double stranded DNA denatures (or melts) in a buffer solution.        Thus, in cases where the nucleic acid sample comprises double        stranded DNA in a buffer solution, the denaturing electrical        potential should be strong enough to apply a stretching force of        at least 65 pN along the DNA strands.

The vcPCR methods of the present invention may be used to effect rapiddenaturation of double stranded nucleic acids without the need for theuse of chemical initiators, accelerators or modifiers of the typedescribed in U.S. Pat. No. 6,365,400 (Stanley). Accordingly, it is to beappreciated that any of the examples or embodiments described hereinmay, in at least some embodiments, be carried out in the absence ofsubstantial amounts of chemical initiators or accelerator compositionsof the type described in U.S. Pat. No. 6,365,400 (Stanley).

Methods for Performing vcPCR

A. Bridge Amplification

In vcPCR Bridge Amplification, both forward and reverse primers arerandomly attached at their 5′ end onto a solid substrate. Following PCRamplification, both forward and reverse strands of the amplifiedtemplates section remain bound at their 5′ ends in a double stranded“bridge” arrangement (FIG. 3).

FIG. 4 shows a vcPCR scheme for bridge amplification. The steps forvcPCR cycling is illustrated in FIG. 4 and is as follows:

-   -   (1) Capture ssDNA from buffer solution onto the forward primer        attached on the insulator oxide (typically Silicon Dioxide) film        on the PCR cell substrate (typically Silicon);    -   (2) Introduce qPCR master mix (Polymerase, dNTP's, buffer, SYBR        Green intercalating dye) into PCR cell to extend forward primer        and make complementary strand (typically 6 seconds or shorter        depending on the polymerase used).    -   (3) Apply a positive voltage on the upper electrode to create a        sufficiently high E-field (in excess of 106 μm for a 100 base        DNA strand) to denature dsDNA and eliminate the target strand.        In our experiments, we applied a field of 4×107 μm.    -   (4) Anneal extended ssDNA complement onto reverse primer by        reducing the applied voltage to zero.    -   (5) Polymerase extension of reverse primer to make dsDNA by        keeping applied voltage at zero for 6 seconds—100 base amplicon        will be extended in 6 seconds for Taq polymerase at a polymerase        extension speed of 1000 bases per minute. Depending on the        specific polymerase used, this speed can be increased by 50×.    -   (6) Apply a positive voltage on the upper electrode (as in        step 3) to denature dsDNA.    -   (7) Anneal melted strands onto new set of forward and reverse        primers by reducing applied voltage to zero.    -   (8) Extend forward and reverse primers to make two double        stranded DNA's by keeping applied voltage at zero for 6 seconds        (for a 100 base amplicon).    -   (9) Go to step (6) to denature the two dsDNA's and repeat        steps (6) to (8) for each PCR cycle.

The total time for 40 PCR cycles is approximately 240 seconds or 4minutes. Amplification after each cycle can be quantified by measuringthe fluorescence level from each cell after excitation with a suitablepump light source as in conventional q PCR.

B. Solid Phase Amplification with a Single Forward Primer

This scheme is essentially similar to scheme (a) except that only theforward primer is attached to the solid surface—just like in emulsionbead PCR; the reverse primer is in the PCR master mix. Thisamplification scheme for vcPCR cycling is illustrated in FIG. 5. Thismethod comprises the following steps:

-   -   (1) Single strand target DNA in buffer captured onto forward        primer on cell substrate surface.    -   (2) qPCR mixture (polymerase, dNTP, buffer, SBR Green        intercalating dye)+reverse primer introduced to extend forward        primer to make complementary strand.    -   (3) Denaturing of dsDNA achieved by applying +X volt on first        (top) electrode to eliminate target strand.    -   (4) Anneal extended ssDNA complement onto reverse primer by        reducing applied voltage to zero.    -   (5) Polymerase extension of reverse primer to make double        stranded DNA by keeping    -   (6) Denaturing of double stranded DNA by applying +X volt to        first (top) electrode.    -   (7) Anneal melted strands onto new set of forward primers by        reducing applied voltage to zero. Anneal reverse primer to        extended forward primer.    -   (8) Polymerase extension of annealed forward and reverse primers        to make two double strand pairs by keeping applied voltage at 0        for 6 seconds.        Devices for Performing vcPCR

FIG. 6 shows an example of a vcPCR device having rectangular wells andFIG. 7 shows an example of a vcPCR device having cylindrical wells. Itis to be appreciated, however, that various other shapes of cells otherthan rectangular or cylindrical may be used. Each of these devicesgenerally comprises a) a base electrode member, b) a gasket member thatis disposed on top of the base electrode member and has openings formedin it to define one or more the sample wells and c) a top electrodemember disposed on top of the gasket member. As shown the gasket memberis positioned on the base electrode member such that at least part ofthe floor of each sample well is formed by the base electrode member.Voltage cycling apparatus may be connected to one or both of said baseand top electrode members to intermittently create an electrical fieldto denature nucleic acid contained within each of the sample well(s). Inthese examples, the voltage cycling apparatus is operative to cerate adenaturing electrical potential is in excess of 10⁶ v/m, for exampleapproximately 4×10⁷ μm, within each of the sample well(s).

The base electrode member may comprise an electrically conductive layerhaving an insulating film disposed thereon. For example, theelectrically conductive layer may comprise doped silicon or anothersuitable electrically conductive material and the insulating film maycomprise silicon dioxide or another suitable insulating film material.

The gasket member may comprise a substantially non-porous electricallyinsulating material such as, for example, a glass, silicon polymer,silicon nitride or Polydimethylsiloxane (PDMS).

The top electrode member may comprises a layer of metal, for example anAu/Cr or Au/Pt film.

In some embodiments, as described above, an oligonucleotide primer maybe attached to a substrate that comprises or is located in proximity tothe base electrode member. For example, the 5′ end of an oligonucleotideprimer may be attached to an insulating film that is attached to thebase electrode member.

The following are typical, but non-limiting, dimensions for the vcPCRdevices of the present invention:

-   -   Electrodes: Cr 10 nm, Au 100 nm or Cr 10 nm, Pt 100 nm    -   Films: Si02 20 nm, ShN4 500 nm    -   Diameter (if cylindrical) or width (if rectangular) of the        sample wells: 1 to 3 fA.m    -   PDMS gasket dimensions: Approximately 2 mm×2 mm-3 mm×3 mm range

Typically, in these exemplary devices, the applied voltage X is in therange of 10-20 volts, but any workable voltage capable of causing thedescribed effects may be used.

The following is a calculation of expected electrical potential andstretching force on an attached DNA double strand

-   -   For X=10 v, assuming sub-wells are filled with buffer (E=36)    -   Electrical potential at the surface of Si02 (E=4),        thickness=0.02 fA.m is    -   E=10V/(0.5+0.02×36/4) fA.m or 1.47×107 Vim    -   Force on a 100 base long ds DNA molecule attached to the Si02        surface is    -   F=100 q E where q is charge of a single base ˜e the electronic        charge so    -   F=882 pN or more than IO× the melting threshold force published        in the literature 1.

FIG. 6 shows fluorescence from 1 μm cylindrical wells after 40 cycles ofvcPCR.

It is to be appreciated that the invention has been described hereabovewith reference to certain examples or embodiments of the invention butthat various additions, deletions, alterations and modifications may bemade to those examples and embodiments without departing from theintended spirit and scope of the invention. For example, any element orattribute of one embodiment or example may be incorporated into or usedwith another embodiment or example, unless otherwise specified of if todo so would render the embodiment or example unsuitable for its intendeduse. Also, where the steps of a method or process have been described orlisted in a particular order, the order of such steps may be changedunless otherwise specified or unless doing so would render the method orprocess unworkable for its intended purpose. All reasonable additions,deletions, modifications and alterations are to be consideredequivalents of the described examples and embodiments and are to beincluded within the scope of the following claims.

1. A method for separating the strands of a double-stranded nucleic acidor double-stranded nucleic acid/primer extension product, said methodcomprising the step of: applying an electrical potential to causenon-thermal separation of strands of the double-stranded nucleic acid ordouble-stranded nucleic acid/primer extension product; wherein theelectrical potential is applied in the substantial absence of lithiumions or any other initiator or promoter substance that a) receiveselectrons from an electrode and transfers them to the double-strandednucleic acid or double-stranded nucleic acid primer extension productand b) has a redox potential of 0 to −2 volts.
 2. A method foramplifying a target nucleic acid sequence contained in a sample thatconsists of or contains one or more nucleic acid(s), said methodcomprising the steps of: a) treating the sample with primers for thetarget sequence under conditions that produce a primer extension productthat is complementary to the target sequence; b) applying an electricalpotential in accordance with the method of claim 1 to separate theprimer extension product from the nucleic acid on which it wassynthesized, thereby producing a single-stranded intermediate product;and c) treating the single-stranded intermediate product with theprimers under annealing conditions to cause a further primer extensionproduct to be produced.
 3. A method according to claim 2 wherein anelectrical potential is also applied during Step a to draw the targetsequence to the primers.
 4. A method according to claim 2 wherein Step bcomprises exposing a reaction mixture that contains at least i) anucleic acid or a mixture of nucleic acids, ii) the primers, iii) apolymerase and iv) extension subunits, to the electrical potential.
 5. Amethod according to claim 4 wherein Step c comprises: terminatingexposure to the electrical potential for a second period of time underconditions that result in annealing of the primers to thesingle-stranded molecules; and allowing or causing polymerase-mediatedproduction of the further extension product.
 6. A method according toclaim 4 wherein: the nucleic acid or mixture of nucleic acids comprisesDNA; the polymerase comprises at least one DNA polymerase; and theextension subunits comprise at least one deoxyribonucleotidetriphosphate.
 7. A method according to claim 6 wherein thedeoxyribonucleotide triphosphate is/are selected from dATP, dTTP, dCTPand dGTP.
 8. A method according to claim 2 wherein at least one of theprimers is attached to a substrate that comprises or is located inproximity to an electrode which, in combination with a counterelectrode,creates the electrical potential in Step b.
 9. A method according toclaim 8 wherein 5′ end(s) of the primer or primers is/are attached tothe substrate.
 10. A method according to any of claims 1-9 wherein theelectrical potential is applied in the substantial absence of anyinitiator or promoter substance selected from the group of: lithiumions, methyl voilogen, benzyl viologen, neutral red, phenosafranin andsalts thereof.
 11. A method according to any of claims 1-9 wherein theelectrical potential is strong enough to apply a stretching force of atleast 65 pN.
 12. A method according to any of claims 2-9 wherein Steps band c are performed at substantially the same temperature.
 13. A methodaccording to claim 12 wherein Steps b and c are carried out at ambienttemperature.
 14. A method according to claim 12 wherein Steps b and care carried out at elevated temperature.
 15. A method according to claim14 wherein the elevated temperature is selected to optimize specificityof the amplified product during annealing.
 16. A method according toclaim 5 wherein the temperature is raised during formation of the primerextension product. 17-43. (canceled)